Analysis of total cytoplasmic and polyadenylated cytoplasmic RNA from cells lytically infected with Simian virus 40 has demonstrated the presence of a small RNA, approximately 65 nucleotides long, which is induced late in lytic infection. This SV40-associated small RNA (SAS-RNA) is apparently specific in size and sequence and is not selected on columns of oligo (dT)-cellulose. It is homologous to a region of the early SV40 mRNAs (and to the late (L)-DNA strand) starting approximately 250 nucleotides from the 3' end of the early mRNAs (SV40 map position 0.21). The function of SAS-RNA in the viral cycle, as well as its source, are unknown at this point; however, its temporal expression, unique sequence and interesting region of homology within the SV40 genome suggest a possible role in the control of SV40 gene expression. In additional experiments, we have attempted to analyze those factors responsible for the relative expression of early and late SV40 mRNA. Among our findings, we noted: 1) the overproduction of early SV40 mRNA and concommitant fall in late transcription which accompanies the shift of a tsA mutant to the nonpermissive temperature, is highly dependent on the monkey cell line. 2) The relative splicing of the primary early SV40 transcript to large-T mRNA and small-t mRNA is strongly influenced by the temperature of the cultures.